Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption
✍ Scribed by R. H. Clemmitt; H. A. Chase
- Publisher
- John Wiley and Sons
- Year
- 2000
- Tongue
- English
- Weight
- 202 KB
- Volume
- 67
- Category
- Article
- ISSN
- 0006-3592
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✦ Synopsis
The facilitated downstream processing of an intracellular, polyhistidine-tagged protein, glutathione Stransferase [GST-(His) 6 ], direct from unclarified E. coli homogenates using expanded beds of STREAMLINE chelating, has been investigated. A series of pilot experiments were used to develop preparative-scale separations of GST-(His) 6 , initially in packed and then in expanded beds. Packed beds of Ni 2+ -loaded STREAMLINE chelating proved to have the highest 5% dynamic capacity for GST-(His) 6 , of 357 U mL -1 (36 mg mL -1 ). When using immobilized Cu 2+ or Zn 2+ , metal ion transfer was observed from the iminodiacetate ligands to the highaffinity chelator, GST-(His) 6 . The subsequent metal affinity precipitation of this homodimer resulted in operational problems. An equilibrium adsorption isotherm demonstrated the high affinity of GST-(His) 6 for immobilized Ni 2+ , with a q m of 695 U mL -1 (70 mg mL -1 ) and a K d of 0.089 U mL -1 (0.0089 mg mL -1 ). Ni 2+ -loaded STREAMLINE chelating was therefore selected to purify GST-(His) 6 from unclarified E. coli homogenate, resulting in an eluted yield of 80% and a 3.34-fold purification. The high dynamic capacity in the expanded mode of 357 U mL -1 (36 mg mL -1 ) demonstrates that this specific interaction was not affected by the presence of E. coli cell debris.